Numerous sources suggest rhodamine 123 as the best probe for the study of ETC modulation using a fluorescent platereader. Is this a suitable method? I've also read that MDR transporters can target R123 and it might be a good idea to block them with verapamil or cyclosporinH to avoid the efflux of R123. Any insight on this control and suitable concentrations? Thanks!
Dear Carlos,
It depends on what kind of modulation of the ETC you want to see? If you want to observe dynamic changes in mitochondria membrane potential, which is of course tightly linked to ETC function, then yes it is a good method. For this a final concentration of 10ug/mL should work well as a dequench mode concentration(30 min incubation at RT). You could use verapamil in order to block MDR transporters but I don`t think uptake into mitochondria will be severely affected at this concentration. Are you limited to a plate reader assay or could you use microscopy or polarographic oxygen consumption measurements? Hope it helps!
Dear Vassilios,
Thanks for your comments. I wasn't sure whether MDR transport could be an issue but it sounds like it should be okay to work around that. I'd initially try to use a plate reader for the sake of simplicity, although I could potentially use fluorescence microscopy too. Cheers!
In isolated mitochondria you mean? is pretty tricky to get it to work in a fluorescent plate reader, you need agitation or you'll get an artefact. However, if you have isolated mitochondria just measure O2 consumption.
In cells use JC1, is a lot more easy to setup. Forget about microscopy, you'll photo-beach Rho123 just for focusing.
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